Identification of Peroxidase Isoenzymes in Satureja hortensis L. Leaves

Satureja hortensis L. is a renowned medicinal plant used in Iranian folk medicine as pain reliever and as treatment for stomach and intestinal disorders. Although agricultural and pharmaceutical properties of this plant have been studied, its enzymatic properties remain unknown. In this study, peroxidase activity in leaf extract was assayed by monitoring spectrophotometrically the H2O2-mediated oxidation of o-dianisidine at 460 nm using an extinction coefficient of 11.3 mM(-1).cm(-1). pH profile showed an optimum peak at pH 5.5 and a shoulder at pH 6.5. Apparent Km, Vmax and catalytic efficiency (calculated per mg protein in the extract) were, respectively, 0.29 +/- 0.03 mM, 47 +/- 2 mu M. min(-1).mg prot(-1) and 0.163 +/- 0.022 min(-1). mg prot(-1) at pH 5.5, 0.6 +/- 0.04 mM, 69 +/- 3 mu M.min(-1).mg prot(-1) and 0.115 +/- 0.014 min(-1). mg prot(-1) at pH 6.5 for o-dianisidine and 2.1 +/- 0.1 mM, 111 +/- 5 mu M.min(-1).mg prot(-1) and 0.053 +/- 0.005 min(-1).mg prot(-1) at pH 5.5, 0.32 +/- 0.02 mM, 58 +/- 3 mu M.min(-1).mg prot(-1) and 0.181 +/- 0.022 min(-1). mg prot(-1) at pH 6.5 for H2O2. The activity was lost after 5 min at 75 degrees C but was still 100% detectable at pH 5.5 after 5 min at 50 degrees C, and at pH 6.5 after 5 min at 40 degrees C. It was sensitive to potassium cyanide and sodium azide, with IC50 of 0.7 mu M at pH 5.5 and 1.5 mu M at pH 6.5 for potassium cyanide, and of 0.6 mM at pH 5.5 and 1 mM at pH 6.5 for sodium azide. Activity staining after electrophoresis of leaf extract in non-denaturing polyacrylamide gel showed three bands, indicating the presence of at least three peroxidase isoenzymes that utilized o-dianisidine as the electron donor substrate.